Silent marker for an oil product and associated detection method

ABSTRACT

A silent marker, method of marking a petroleum product with the silent marker, and method of detecting the silent marker. The marker is an ester derivative of fluorescent material, and the silent marker may be detected by measuring the fluorescence generated from the selective hydrolysis of the ester moiety under enzymatic action.

This application is filed in accordance with 35 U.S.C. 371 as a NationalStage Application of PCT/KR01/01202, filed Jul. 12, 2001, which claimspriority to Applications KR 2000/0039815, filed Jul. 12, 2000, and KR2001/0032365, filed Jun. 9, 2001.

TECHNICAL FIELD

The present invention relates to marking a petroleum product with asilent marker and to the detection of the silent marker. Moreparticularly, the present invention is directed to a method for markinga petroleum product with a silent marker which is ester derivative offluorescent material and a method for detecting the silent marker bymeasuring the fluorescence generated therefrom by use of enzymatichydrolysis.

PRIOR ART

Recently, the sale of counterfeit petroleum products has beendramatically increased for a variety of reasons, for example, governmenttax policies, the rising price of raw materials for petroleum products,and the like. Further, when considering the serious effects ofcounterfeit fuels on the life span of vehicles and the environment, itis required to prevent the use thereof. However, since it is verydifficult to identify such counterfeit petroleum products with the nakedeye, complicated chemical analyses should be carried out.

The term “fluorescent material” as used herein is defined as a substancewhich, when irradiated with light of specific wavelength, generateslight with a different wavelength from that of the applied light byforming an electronic resonance structure. As such, even though afluorescent material is present at a small amount in petroleum products,it may be detected with ease by irradiating visible light or ultravioletthereto. However, most fluorescent materials have poor solubility in thepetroleum products such as gasoline.

Herein, “marker” means a substance in which a C₄-C₁₈ hydrocarbon groupis attached to a reactive site of the fluorescent material throughesterification. The marker as mentioned above show high solubility inthe petroleum products, and do not exhibit fluorescence themselves sincethey have lost the electronic resonance structure owing toesterification. However, when the ester moiety is decomposed therefrom,the fluorescent material is so soluble in water as to re-exhibitfluorescence. Thus, if petroleum products are marked with such a markerprior to distribution, it can be confirmed whether or not the genuinepetroleum products are replaced and/or blended with low priced productsmanufactured by another company, or low-grade products, byquantitatively analyzing the fluorescence of the marker in the petroleumproducts on distribution network. Further, the lightly taxed productsmay be identified from the similar products subject to higher taxes inthe same way as explained above.

In this regard, U.S. Pat. No. 5,980,593 discloses linear or branchedC₁-C₁₈ alkyl acid ester 7-hydroxy-4-methylcoumarin as a silentfluorescent material for marking petroleum products. In the conventionaltechniques, including the above patent, chemical methods have beenemployed for the identification of the silent marker, in which the estermoiety is decomposed using alkaline solution. However, the fluorescentmaterial may be destroyed during the above procedure, which makes itvery difficult to analyzing the fluorescent material quantitatively.Thus, the fluorescent materials available for the conventional methodsare considerably limited. Furthermore, other fluorescent materials,which are present naturally in petroleum products, can be extracted byalkaline solution along with the desired fluorescent materials, therebypreventing accurate quantitative analysis of the silent marker.

In particular, additional heating equipments and catalysts are requiredto carry out the above-mentioned procedures. Thus, it not only takes along time to detect fluorescence, but also it lacks practicaleffectiveness. Also, the ester moiety is relatively stable, so thatother moieties which are less stable than ester moiety are decomposed bythe alkaline solution. As a result, the silent marker employable in theconventional methods is limited to the specific ones, for instancemarker as disclosed in the above patent.

DESCLOSURE OF THE INVENTION

It is an object of the present invention to provide a silent marker,which is ester derivative of fluorescent material, suitable for markinga petroleum product.

It is another object of the present invention to provide a compositionfor detecting a silent marker, which includes an enzyme or apolymer-linked enzyme capable of selectively hydrolyzing the estermoiety of the silent marker.

It is a further object of the present invention to provide a silentmarking method for a petroleum product.

It is a further object of the present invention to provide a detectionmethod of a silent marker for a petroleum product using an enzyme or apolymer-linked enzyme capable of selectively hydrolyzing the estermoiety of the silent marker.

It is still another object of the present invention to provide a methodfor identifying a petroleum product through enzymatic action.

In accordance with first aspect of the present invention, there areprovided silent markers represented by the following Formulas I, II andIII:

wherein, R₁ and R₂, which are the same or different, are a C₄-C₁₈ alkylgroup.

wherein, R₃ is a C₄-C₁₈ alkyl group; R₄ is a hydrogen atom, methyl groupor trifluoromethyl group; and R₅ is a hydrogen atom, a halogen atom orcyano group.

wherein, R₆ and R₇, which are the same or different, are a C₁₉-C₂₄ alkylgroup.

In accordance with the second aspect of the present invention, there isprovided a silent marking method for a petroleum product, in which amarker is added to the petroleum product, said marker being an esterderivative of fluorescent material.

In accordance with the third aspect of the present invention, there isprovided a detection method of a silent marker for a petroleum product,comprising the steps of:

a) marking the petroleum product with the silent marker, which is anester derivative of fluorescent material;

b) adding the petroleum product with an detection composition containingan enzyme capable of specifically decomposing the ester moiety of thesilent marker; and

c) measuring the fluorescence generated from the step b).

In accordance with the fourth aspect of the present invention, there isprovided a method for identifying a petroleum product, comprising thesteps of:

a) adding the petroleum product having been marked with a silent marker,which is an ester derivative of fluorescent material, with a compositioncontaining an enzyme capable of specifically decomposing the estermoiety of the marker; and

b) measuring the fluorescence generated from the step a).

In accordance with the fifth aspect of the present invention, there isprovided a composition for detection of a silent marker, which is anester derivative of fluorescent material, in a petroleum product,wherein said composition comprises an aqueous solution of pH 7 or highercontaining an enzyme capable of selectively hydrolyzing the ester moietyof the marker.

BRIEF DESCRIPTION OF THE DRAWINGS

The above objects, and other features and advantages of the presentinvention will become more apparent from the following detaileddescription when taken in conjunction with the drawings, in which:

FIG. 1 is a graph illustrating the quantitative analysis of petroleumfor resorufin dipalmitate content up to 100 ppb by use of a potablefluorometer according to Example 12 of the present invention;

FIG. 2 is a graph illustrating the quantitative analysis of petroleumfor fluoresceine dipalmitate content up to 100 ppb by use of a potablefluorometer according to Example 13 of the present invention;

FIG. 3 is a graph illustrating the quantitative analysis of petroleumfor 3-choro-4-methyl-7-hydroxy coumarin palmitate content up to 100 ppbby use of a potable fluorometer according to Example 14 of the presentinvention;

FIG. 4 is a graph illustrating the quantitative analysis of petroleumfor a fluorescent material excited with light of wavelength of 350 nm byuse of a spectrofluorometer according to Example 20 of the presentinvention; and

FIG. 5 is a graph illustrating the relationship between the luminescenceand the concentration of the silent marker according to Example 19 ofthe present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

In accordance with the present invention, the silent marker refer to anester derivative of fluorescent material, in which a hydrocarbon groupis attached to a reactive site such as a hydroxyl group, of thefluorescent material, through esterification. There are no limitationsas to the silent marker employable herein, with the proviso that saidsilent marker is capable of regaining fluorescence through hydrolyzingthe ester moiety thereof. In particular, alkyl acid esters of resorufin,fluorescein or coumarin may preferably be used as a silent marker.

The silent markers in accordance with the present invention may berepresented by the following Formulas I, II and III;

wherein, R₁ and R₂, which are the same or different, are a C₄-C₁₈ alkylgroup. Preferably, each of R₁ and R₂ is a C₁₅H₃₁ group.

wherein, R₃ is a C₄-C₁₈ alkyl group; R₄ is a hydrogen atom, methyl groupor trifluoromethyl group; and R₅ is a hydrogen atom, a halogen atom orcyano group. Preferably, R₃ is a C₁₅H₃₁ group, R₄ is a methyl group, andR₅ is a chlorine atom.

wherein, R₆ and R₇, which are the same or different, are a C₁₉-C₂₄ alkylgroup. Especially, R₆ and R₇ serve to increase the solubility of thesilent marker of Formula III in organic liquids.

In the present invention, representatives of said silent marker includeester derivatives of 7-hydroxy-4-trifluoromethyl coumarin, 7-hydroxycoumarin, 3-cyano-7-hydroxy-4-methyl coumarin, 4-hydroxy coumarin,7-hydroxyl-4-methyl coumarin (U.S. Pat. No. 5,980,593),3-chloro-7-hydroxy-4-methyl coumarin and 3-chloro-7-hydroxy-4-methylcoumarin, ester derivatives of resorufin, ester derivatives offluorescein (U.S. Pat. No. 5,498,808), and a mixture thereof.

According to the present invention, as the petroleum product to whichthe above marker is added, there may be used fuel for vehicles such asgasoline, liquefied natural gas (LNG), liquefied petroleum gas (LPG),diesel fuel, kerosene, heavy oil, and the like. Thus, petroleum productsfor various uses, which are manufactured and/or distributed by taxevaders, as well as counterfeit petroleum products prepared by mixingorganic solvents such as benzene, toluene and the like, can beidentified with ease.

In the present invention, the marker may be used at an amount of 0.01-30ppm, preferably, 0.5-10 ppm in the petroleum product. When detected, thefluorescent material corresponding to the silent marker releases aunique fluorescence wavelength from one another. In the case where thepetroleum product is marked using a mixture of two kinds of markers,which are mixed with each other at known ratio, the third fluorescenceresulting from the combination of two different fluorescences may bedetected. In this regard, for example, it is possible to mark gasolineand kerosene with different silent markers.

Furthermore, since the silent marker is added to the petroleum productat the predetermined amount, it can be detected so quantitatively thatan amount of the marked petroleum product in the sample can bedetermined. That is, if the marker is added to the genuine petroleumproduct at a constant amount, it is possible to confirm not only whetheror not the petroleum product is genuine by observing the developedfluorescence, but also whether the petroleum product is blended withother liquids by measuring the intensity of the fluorescence.

According to the present invention, in order to detect the marker, thereis used an enzyme capable of selectively hydrolyzing the ester moiety ofthe marker. In particular, an aqueous solution (pH 7 or higher)containing a constant concentration of enzyme is dispensed into adetection kit at a constant amount and then lyophilized to increase theaccessibility to organic liquids and the reproductivity of the reactionfor detection. There is disclosed the above method for improving thereproductivity of the reaction using a lyophilized enzyme in KoreanPatent No. 162270.

Preferably, an enzyme suitable for the present invention is selectedfrom the group consisting of lipase, esterase and cellulase, which maybe obtained from various microorganisms or from the internal organs ofanimals. Most of these enzymes work at a relatively high temperature,for example 50° C. or higher, and have such excellent heat stabilitythat the composition for detecting the marker can be stored with ease.Among them, lipase is more stable in organic liquids than other enzymes,since it acts in vivo as a digestive enzyme for fat.

Further, the enzyme may be used with a (or one or more) polymeric moiety(hydrocarbon polymeric moiety) linked thereto in order to increase bothsolubility and stability of the enzyme in organic liquids. Preferably,this hydrocarbon polymer(s) to be linked to the enzyme has a molecularweight of 2000 Daltons or higher, and polyethyleneglycol (PEG), Triton Xand the like can be preferably used with more preference.

For linking the enzyme to the polymeric moiety, a compound containingboth a portion capable of reacting with amine groups, sulfhydryl groups,or hydroxyl groups in amino acids; and a portion capable of reactingwith hydroxyl groups in the polymer, can be preferably used as a linker.The exemplified structure in which the enzyme is linked to polymericmoieties by the linker such as cyanuric chloride, is represented by thefollowing Formula IV:

In the linking reaction between the enzyme and the polymer, a lysineresidue, which exists on the surface of the enzyme, may be linked to thepolymer at a ratio of either 1:1 or 2:1, since the mixing ratio betweenthe enzyme and the polymer varies with the number of lysine residues onthe enzyme.

In the case where the enzyme linked to the polymeric moiety is used forthe detection of the marker, there are provided the followingadvantages.

Firstly, proteases, which may deactivate the enzyme, can be preventedfrom accessing the enzyme. Secondly, the environment in which the enzymeforms an active three-dimensional structure through hydrogen bonds withwater molecules is also established within organic solvents such thatthe enzyme activity can be greatly improved therein. Thirdly, enzymeactivity is conserved for over 3 months even though the enzyme is storedin organic solvent at room temperature, since the destruction of thethree dimensional structure of the enzyme, which is caused by exposureof the hydrophobic portion of enzyme to the solvent as the temperatureincreases (Inada, Y. et al., 1986), is prevented by the hydrocarbonpolymeric moiety.

Aqueous solution (pH 7 or higher), which contains the enzyme linked topolymeric moiety described above, can be used as the composition ofdetection. Preferably, the above aqueous solution is used afterlypophilization.

The above aqueous solution is added to the petroleum product marked witha silent marker at a proper ratio and mixed well. Then, the resultantaqueous solution layer is irradiated with light of one specificwavelength, so that light of another specific wavelength is releasedtherefrom. In this case, most lights which are fluoresced from theaqueous solution layer are visible light. For instance, resorufinreleases a red light, fluorescein releases a green light, and coumarinreleases a blue light. The fluorescence ability results from hydrolysisof ester bond of the silent marker by use of the ester-decomposableenzyme. The enzyme reaction occurs in the phase boundary betweenpetroleum and water, since enzyme in aqueous solution uses water todecompose the ester bond of the marker dissolved in the petroleum phase,and then fluorescent material, which is dissociated from the hydrocarbonmoiety of the marker, is released into the water phase. Thus, thefluorescence generated from the water phase can be detected by use ofspectrophotofluorometer. Also, where any portablespectrophotofluorometer is used, fluorescence in the petroleum productcan be detected easily with precision.

Hereinafter, the present invention will be described in detail withreference to the following examples. The examples are given forillustration of the present invention and are not to be construed aslimiting the present invention.

EXAMPLE 1 Synthesis of Resorufin Dipalmitate

1.6 g of resorufin was dissolved in 40 ml of tetrahydrofuran in a 250 mlflask, and then 3.1 g of potassium carbonate and 3.1 ml of triethylaminewere added to the solution. After 30 minutes, 7.0 ml of palmitoylchloride was added thereto and the mixture was stirred for 2 hours atroom temperature. Thereafter, 50 ml of distilled water and 250 ml ofethyl acetate were added to extract the title compound into the ethylacetate phase. The ethyl acetate phase was washed with 200 ml of saltwater, and then dried with manganese anhydride. Following thedistillation and evaporation of the solvent, the residue wasrecrystalized with hexane and ethyl acetate to obtain reddish resorufindipalmitate.

EXAMPLE 2 Synthesis of Fluorescein Dipalmitate

2.5 g of fluorescein was dissolved in tetrahydrofuran in a 250 ml flask,and then 3.1 g of potassium carbonate and 3.1 ml of triethylamine wereadded to the solution. After 30 minutes, 7.0 ml of palmitoyl chloridewas added thereto and the mixture was stirred for 2 hours at roomtemperature. Next, 50 ml of distilled water and 250 ml of ethyl acetatewere added to extract the title compound into the ethyl acetate phase.The ethyl acetate phase was washed with 200 ml of salt water, and thendried with manganese anhydride. Following the distillation andevaporation of the solvent, the residue was recrystalized with hexaneand ethyl acetate to obtain fluorescein dipalmitate of the color ofskin.

EXAMPLE 3 Synthesis of 3-chloro-4-methyl-7-hydroxy Coumarin Palmitate

2.5 g of 3-chloro-4-methyl-7-hydroxy coumarin was dissolved in 60 ml oftetrahydrofuran in a 250 ml flask, and then 3.6 g of potassium carbonateand 3.6 ml of triethylamine were added to the solution. After 30minutes, 10.0 ml of palmitoyl chloride was added thereto and the mixturewas stirred for 2 hours at room temperature. Thereafter, 50 ml ofdistilled water and 250 ml of ethyl acetate were added to extract thetitle compound into the ethyl acetate phase. The ethyl acetate phase waswashed with 200 ml of salt water, and then dried with manganeseanhydride. Following the distillation and evaporation of the solvent,the residue was recrystalized with hexane and ethyl acetate to obtainwhite 3-chloro-4-methyl-7-hydroxy coumarin palmitate.

EXAMPLE 4 Synthesis of 7-hydroxy Coumarin Palmitate

1.0 g of 7-hydroxy coumarin was dissolved in 100 ml of pyridine in a 500ml flask and 2.9 ml of palmitoyl chloride was added to the solution,followed by stirring at room temperature. After reaction for 3 hours,pyridine was removed by evaporation under vacuum. Addition of distilledwater and methylene chloride at ratio of 1:1 allowed the title compoundto be extracted into the methylene chloride phase. The methylenechloride phase was subjected to chromatography eluting with methylenechloride only. The solvent was removed from the first eluate to obtainwhite 7-hydroxy coumarin palmitate.

EXAMPLE 5 Synthesis of 3-chloro-4-methyl-7-hydroxy Coumarin Laurate

2.5 g of 3-chloro-4-methyl-7-hydroxy coumarin was dissolved in 60 ml oftetrahydrofuran in a 250 ml flask, followed by the addition of 3.6 g ofpotassium carbonate and 3.6 ml of triethylamine. After 30 minutes, 8.0ml of lauroyl chloride was added thereto and the mixture was stirred for2 hours at room temperature. Thereafter, 50 ml of distilled water and250 ml of ethyl acetate were added to extract the title compound intothe ethyl acetate phase. The ethyl acetate phase was washed with 200 mlof salt water, and then dried with anhydrous manganese. Following thedistillation and evaporation of the solvent, the residue wasrecrystalized with hexane and ethyl acetate to obtain white3-chloro-4-methyl-7-hydroxy coumarin laurate.

EXAMPLE 6 Preparation of Reagent Containing Lipase for Detection

At 4° C., 1 g of the lipase purified from Pseudomonas cepacis wascompletely dissolved in 25 ml of a 100 mM Tris buffer adjusted to pH 8.0with hydrochloric acid. After being dispensed by 20 ul into 2 ml tubes,the solution was dried on Centra evaporator™ (Bioneer Corporation,Korea) and stored frozen.

EXAMPLE 7 Preparation of Lipase Linked to Polyethyleneglycol

20.00 g of monomethoxypolyethyleneglycol (M.W. about 4,500) wasdissolved, along with 0.37 g of cyanuric chloride as a linker, in 100 mlof benzene in a 500 ml flask. After the addition of 6.5 g of NaCO₃thereto, the reaction was conducted at 80° C. for 40 hours. Prepared bysubstituting a methoxy group for one of the two terminal hydroxyl groupsof polyethylene glycol, the monomethoxypolyethyleneglycol is ahydrocarbon polymer containing only one active hydroxyl group. Saltswere filtered off and the solvent was removed to give white solids. 5 gof the solids were dissolved, along with 78 mg of a lipase purified fromPseudomonas cepacis, in 25 ml of a 100 mM Tris buffer adjusted to pH 8.0with hydrochloric acid, and the reaction was carried out for 2 hours at4° C. After the removal of unreacted polyethylene glycol by filtrationwith a 1 kDa cutoff, the solution was dispensed into 1.5 ml tubes by 20μL, dried on a Centra evaporator™ (Bioneer Corporation, Korea) andstored frozen. This was used as a detecting composition in subsequentexperiments.

EXAMPLE 8 Qualitative Detection of Resorufin Dipalmitate in Petroleum

To the enzyme solution for detection obtained in Example 6 were added0.5 ml of distilled water and 1.0 ml of petroleum containing 1 ppm ofthe resorufin dipalmitate obtained in Example 1, followed by stirringthe solution. When the aqueous phase was irradiated with natural light,red light was detected by the naked eye.

EXAMPLE 9 Qualitative Detection of Fluorescein Dipalmitate in Petroleum

To the enzyme solution for detection obtained in Example 6 were added0.5 ml of distilled water and 1.0 ml of petroleum containing 1 ppm offluorescein dipalmitate obtained in Example 2, followed by stirring thesolution. When the aqueous phase was irradiated with natural light,green light was detected by the naked eye.

EXAMPLE 10 Qualitative Detection of 3-chloro-4-methyl-7-hydroxycoumarinPalmitate in Petroleum

To the enzyme solution for detection obtained in Example 6 were added0.5 ml of distilled water and 1.0 ml of petroleum containing 10 ppm of3-chloro-4-methyl-7-hydroxycoumarin palmitate obtained in Example 3,followed by stirring the solution. When the aqueous phase was irradiatedwith light of wavelength of 365 nm, blue light was detected by the nakedeye.

EXAMPLE 11 Qualitative Detection of a Mixture of Resorufin Dipalmitateand 3-chloro-4-methyl-7-hydroxycoumarin Palmitate in Petroleum

Resorufin dipalmitate and 3-chloro-4-methyl-7-hydroxycoumarin palmitateobtained in Examples 1 and 3 were added to petroleum in amounts of 1 and10 ppm, respectively. To the enzyme solution obtained in Example 6 wereadded 0.5 ml of distilled water and 1.0 ml of petroleum containing themarkers, followed by stirring the solution. When the aqueous phase wasirradiated by light of wavelength of 365 nm, violet light was detectedby the naked eye.

EXAMPLE 12 Quantitative Detection of Resorufin Dipalmitate in Petroleum

Kerosene containing 100 ppb (w/v) of the marker obtained in, Example 1was diluted by 0/100, 1/100, 2/100, 10/100, 20/100 and 100/100 to givesolutions of 0 ppb, 1 ppb, 2 ppb, 5 ppb, 10 ppb, 20 ppb, 50 ppb and 100ppb respectively, each of which was then dispensed by 1 ml to the enzymesolution obtained in Example 6, followed by mixing with 1.5 ml ofdistilled water. While the aqueous phase was irradiated with light ofwavelength of 570 nm, the fluorescent light at 585 nm was quantitativelymeasured by use of a fluorometer, and the result is depicted in FIG. 1.

EXAMPLE 13 Quantitative Detection of Fluorescein Dipalmitate inPetroleum

Petroleum containing 100 ppb(w/v) of the marker obtained in Example 2was diluted by 0/100, 1/100, 2/100, 5/100, 10/100, 20/100, 50/100 and100/100 to give solutions of 0 ppb, 1 ppb; 2 ppb; 5 ppb; 10 ppb; 20 ppb,50 ppb and 100 ppb, respectively, each of which was dispensed by 1 mlinto the enzyme solution obtained in Example 6, followed by mixing with1.5 ml of distilled water. The aqueous phase was irradiated with lightof wavelength of 490 nm, the fluorescent light at 514 nm wasquantitatively measured by use of a fluorometer, and the result isillustrated in FIG. 2.

EXAMPLE 14 Quantitative Detection of 3-chloro-4-methyl-7-hydroxycoumarinPalmitate in Petroleum

Petroleum containing 100 ppb(w/v) of the marker obtained in Example 3was diluted by 0/10, 1/10, 2/10, 5/10 and 10/10 to give solutions of 0ppb, 10 ppb, 20 ppb, 50 ppb, 100 ppb, respectively, each of which wasdispensed by 1 ml into the enzyme solution obtained in Example 6,followed by the mixing 1.5 ml of distilled water. The aqueous phase wasirradiated with light of wavelength of 350 nm, the fluorescent light at470 nm was quantitatively measured by use of a fluorometer, and theresult is illustrated in FIG. 3.

EXAMPLE 15 Quantitative Detection of 4-methyl-7-hydroxycoumarinPalmitate in Petroleum

Petroleum containing 100 ppb(w/v) of 4-methyl-7-hydroxycoumarinpalmitate was diluted by 0/10, 1/10, 2/10, 5/10 and 10/10 to givesolutions of 0 ppb, 10 ppb, 20 ppb, 50 ppb, and 100 ppb, respectively,each of which was dispensed by 1 ml into the enzyme solution obtained inExample 6, followed by mixing with 1.5 ml of distilled water. Whileaqueous phase was irradiated with light of wavelength of 365 nm, thefluorescent light at 470 nm was quantitatively measured by use of afluorometer.

EXAMPLE 16 Qualitative Detection of Fluorescein Dipalmitate in Petroleum

To the enzyme solution obtained in Example 7, distilled water andpetroleum containing 10 ppm of fluorescein palmitate obtained in Example2 were added in the same amount, followed by stirring. When the aqueousphase was irradiated with ultraviolet light of wavelength of 365 nm,blue light was detected by the naked eye.

EXAMPLE 17 Qualitative Detection of 3-chloro-4-methyl-7-hydroxycoumarinPalmitate in Petroleum

To the enzyme solution obtained in Example 7, distilled water andpetroleum containing 10 ppm of 3-chloro-4-methyl-7-hydroxycoumarinpalmitate of Example 3 were added in the same amount, followed bystirring. When the aqueous phase was irradiated with ultraviolet lightof wavelength of 365 nm, the blue light was detected by the naked eye.

EXAMPLE 18

Qualitative Detection for Petroleum Containing a Mixture of FluoresceinDipalmitate and 3-chloro-4-methyl-7-hydroxycoumarin Palmitate

In petroleum were dissolved fluorescein dipalmitate and3-chloro-4-methyl-7-hydroxycoumarin palmitate, which were obtained inExample 2 and 3, respectively. To the enzyme solution obtained, inExample 7, distilled water and the petroleum containing the markers asabove were added in the same amount, followed by stirring. When theaqueous phase was irradiated with ultraviolet light of wavelength of 365nm, yellowish light was detected by the naked eye.

EXAMPLE 19 Quantitative Detection of Fluorescein Dipalmitate inPetroleum

Petroleum containing 250 ppm(w/v) of fluorescein palmitate obtained inExample 2 was diluted by 1, 1/2, 1/4, 1/8, 1/16 and 1/32 to givesolutions of 250 ppm, 125 ppm, 62.5 ppm, 31.3 ppm, 15.6 ppm and 7.8 ppmrespectively, each of which was dispensed by 0.5 ml into the enzymesolution obtained in Example 7, followed by mixing with 0.6 ml ofdistilled water. After 0.5 ml of the aqueous phase was diluted by 4folds, fluorescent light emitted from the diluted aqueous phase wasquantitatively measured by use of a spectrofluorometer (JASCO. FP-750).The results are depicted in FIG. 5 in which the intensity of fluorescentlight at 450 nm is linearly plotted versus the concentration of thelabeling material.

EXAMPLE 20 Quantitative Detection of 3-chloro-4-methyl-7-hydroxycoumarinPalmitate

Petroleum containing 250 ppb(w/v) of 3-chloro-4-methyl-7-hydroxycoumarinpalmitate obtained in Example 3 was diluted by 1, 1/2, 1/4, 1/8, 1/16and 1/32 to give solutions of 250 ppm, 125 ppm, 62.5 ppm, 31.3 ppm, 15.6ppm and 7.8 ppm, respectively, each of which was dispensed by 0.5 mlinto the enzyme solution obtained in Example 7, followed by mixing with0.6 ml of distilled water. After the dilution of the aqueous phase by 4folds, fluorescent light emitted from the diluted aqueous phase wasquantitatively measured by use of a spectrofluorometer (JASCO. FP-750).When irradiated with light of wavelength of 350 nm, a maximal peak wasobtained at 470 nm, as shown in FIG. 4.

INDUSTRIAL APPLICABILITY

The present invention has very high sensitivity since enzyme, which canselectively hydrolyze ester moiety, is used to detect the silent markerand background signal by fluorescent material present naturally in thepetroleum product can be greatly reduced. Also, the procedures fordetecting the marker are very simple and easy since only enzymesolution, which can selectively hydrolyze ester bond, is added to thepetroleum product. In addition, the enzyme system of the presentinvention is capable of detecting several markers in petroleum at thesame time.

Where the present invention is applied to genuine petroleum,fluorescence is released only from the genuine petroleum, thus, acounterfeit petroleum can be distinguished from the genuine one.Further, according to the present invention, there is not required acomplicated procedure such as chemical analysis for the detection of themarker, whereby the present invention may be carried out by a consumeron the spot. For example, after petroleum containing a marker and waterare added to the kit containing the dried enzyme solution and shaken, itcan be confirmed whether the petroleum is genuine or not.

1. A method for detection of a silent marker in a petroleum product,comprising the steps of: a) marking the petroleum product with thesilent marker, which is an ester derivative of fluorescent material; b)adding a detection composition to the marked petroleum product, whereinthe detection composition contains an enzyme that specificallydecomposes the ester moiety of the silent marker; and c) detecting thepresence of the silent marker by measuring the fluorescence generatedfrom step b).
 2. The detection method according to claim 1, wherein saidmarker is represented by the following Formula I:

wherein, R₁ and R₂, which are the same or different, are C₄-C₁₈ alkylgroup.
 3. The detection method according to claim 1, wherein said markeris represented by the following Formula II:

wherein, R₃ is C₄-C₁₈ alkyl group; R₄ is methyl or trifluoromethyl; andR₅ is a hydrogen atom, a halogen atom or cyano group.
 4. The detectionmethod according to claim 1, wherein said marker is represented by thefollowing Formula III:

wherein, R₆ and R₇, which are the same or different, are a C₁₉-C₂₄ alkylgroup.
 5. The detection method according to claim 1, wherein saidfluorescent material is selected from the group consisting of esterderivatives of 7-hydroxy-4trifluoromethyl coumarin, 7-hydroxycoumarin,3-cyano-7-hydroxy-4-methyl coumarin, 4-hydroxy coumarin,7hydroxy-4-methyl coumarin and 3-chloro-7-hydroxy-4methyl coumarin,ester derivatives of resorufin, ester derivatives of fluorescein, and amixture thereof.
 6. The detection method according to claim 3, whereinR₄ is methyl and R₅ is a chlorine atom.
 7. The detection methodaccording to claim 2, wherein each of R₁ and R₂ is a C₁₅H₃₁ group. 8.The detection method according to claim 1, wherein said marker is addedin an amount of 0.01-30 ppm to the petroleum product.
 9. The detectionmethod according to claim 1, wherein said petroleum product is selectedfrom the group consisting of gasoline, liquefied natural gas (LNG),liquefied petroleum gas(LPG), diesel fuel, kerosene and heavy oil. 10.The detection method according to claim 1, wherein said enzymeselectively hydrolyses the ester moiety of the marker.
 11. The detectionmethod according to claim 1, wherein said enzyme is selected from thegroup consisting of esterase and cellulase.
 12. The detection methodaccording to claim 1, wherein said enzyme is lipase.
 13. The detectionmethod according to claim 1, wherein said enzyme is linked to at leastone polymeric moiety.
 14. The detection method according to claim 13,wherein said enzyme is linked to the polymeric moiety by a linker. 15.The detection method according to claim 14, wherein said linker iscyanuric chloride.
 16. The detection method according to claim 13,wherein said polymer-linked enzyme is represented by the followinggeneral Formula IV;


17. The detection method according to claim 13, wherein said polymericmoiety is polyethyleneglycol.
 18. A method for identifying a petroleumproduct, comprising the steps of: a) mixing the petroleum product havingbeen marked with a silent marker, which is an ester derivative offluorescent material, with a composition containing an enzyme thatspecifically decomposes the ester moiety of the marker to identify themarked petroleum product; and b) measuring the fluorescence generatedfrom step a).
 19. The method according to claim 18, wherein said enzymeselectively hydrolyses the ester moiety of the marker.
 20. The methodaccording to claim 18, wherein said enzyme is selected from the groupconsisting of esterase and cellulase.
 21. The method according to claim18, wherein said enzyme is lipase.